The mechanism by which herpes simplex virus type-1 (HSV-1) infects the target cell is not completely understood. According to the current model, HSV envelope glycoproteins, gB and gC, initiate contact with host cells by binding to heparan sulfate (HS) proteoglycans. Subsequently, a third glycoprotein D, (gD), binds to one of its receptors. The binding of gD to its receptor triggers fusion of virion envelope with the plasma membrane of the host cell. Previous studies demonstrated that unique sites within HS, or 3-O sulfated heparan sulfate (3-OS HS), generated by an enzyme heparan sulfate 3-O-sulfotransferase isoform-3 (3-OST-3) that can serve as gD receptor for HSV-1. My current research demonstrates that expression of zebrafish (ZF) encoded 3-OST-3 allow HSV-1 entry in resistant CHO-K1 cells, while pre-treatment of ZF embryos with heparinase enzyme significantly blocks HSV-1 entry, further indicating the in vivo significance of this unique enzyme. The studies proposed here will analyze the structural and biochemical aspects of HSV-1 gD and ZF encoded 3-OS HS interactions. Apart from that, our aim is to identify novel anti-3-OS HS peptides to inhibit HSV-1 gD interaction with 3-OST-3 and block viral entry. The selected candidate peptides that block HSV-1 infection in cell culture will be further developed and characterized in ZF embryo model for the development of novel and effective therapeutic strategies. The development of ZF model to study viral entry and spread is innovative and also a first step for their use in screening of inhibitors against HSV. ZF's competitive advantage over other model systems is "optical clarity" in a vertebrate embryo amenable to visualize and track HSV-1 spread in different cells and tissues (by the use of GFP-tagged HSV-1) and large-scale screening of receptor-specific and small viral entry inhibitor molecules. The transparency of ZF embryos and early larvae permits the observation of HSV-1 spread and associated cell or tissue tropism in ZF embryo to be made in vivo on intact animals which are not possible with the existing mouse or rabbit models used to study HSV-1 pathogenesis. A further advantage of using ZF assays over traditional mammalian models is the short duration of such assays. Our recent observation further indicates that cornea of ZF embryo infected with HSV-1 leads to extensive dendrite formation followed by cell damage which mimics the dendrites of herpetic stromal keratitis (HSK) reported in human cornea infected with HSV-1. This result reflects the clinical significance of ZF model to study HSV pathogenesis and an opportunity to understand the role of 3-OS HS in the process of viral entry and spread in embryo model of ZF. The specific aims of this proposal are: AIM 1. Functional analysis of ZF encoded 3-OS HS variants generated by 3-OST isoforms that allows HSV-1 entry. AIM 2. Determine the structural basis for a productive interaction between HSV-1 glycoprotein D and ZF encoded 3-OS HS. AIM 3. Generation of anti-3-OS HS-peptides as novel tools to study gD and 3-OS HS interaction and verify their involvement in the blocking HSV-1 entry and spread in embryo model of ZF. PUBLIC HEALTH RELEVANCE: Herpes Simplex Virus type-1 (HSV-1) is a significant health problem in US. Using embryo model of zebrafish (ZF) we propose to study viral entry and spread since ZF embryo expresses viral entry receptors. Apart from that, our aim is to identify novel peptides to inhibit HSV-1 entry and spread in embryo model of ZF.